Project description:With increasingly concerning strains of antimicrobial resistant strains of the commensal, gram-negative bacteria Klebsiella pneumoniae emerging, there is a pressing need to better understand the pathogen and mechanisms behind its pathogenicity. This study investigated the regulatory mechanisms in strain MGH 78578 of two major sigma factors, the house-keeping sigma factor RpoD, and the general stress response sigma factor RpoS, in mid-exponential and early stationary phase using chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) followed by deep sequencing. Combining ChIP-exo and transcriptome analysis allowed for the determination of sigma factor binding sites, binding motifs, and genes included in the phase-specific sigmulons. The number of genes included in the RpoS sigmulon was greater than in the RpoD sigmulon, with 1,833 and 1,690 genes included, respectively; however, a majority of sigmulon genes were found in all phase-specific sigmulons. Focussing on pathogenicity genes, 20 antimicrobial resistance genes (ARGs) and 155 virulence genes, only two ARGs were found exclusively in one phase-specific sigmulon, an oxacillin-hydrolysing class D beta-lactamase and chloramphenicol efflux MFS transporter CmlA5, which were found in the RpoD sigmulon in early stationary phase. Notably, six unnamed proteins that are or pertain to fimbrial proteins were found uniquely in the RpoS sigmulon in early stationary phase. From this, it can be hypothesised that early stationary phase might be an important phase for pathogenicity gene regulation. While there is little conservation in RpoS sigmulons from strain to strain, RpoS appears to have a consistent overarching role across strains, including a role as a regulator of pathogenicity genes.
Project description:With the global increase in the use of carbapenems, several gram-negative bacteria have acquired carbapenem resistance, thereby limiting treatment options. Klebsiella pneumoniae is one of such notorious pathogen that is being widely studied to find novel resistance mechanisms and drug targets. These antibiotic-resistant clinical isolates generally harbor many genetic alterations, and identification of causal mutations will provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance, in which mutated genes that also show significant expression changes among their functionally coupled genes become more likely candidates. For network-based analyses, we developed a genome-scale co-functional network of K. pneumoniae genes, KlebNet (www.inetbio.org/klebnet). Using KlebNet, we could reconstruct functional modules for antibiotic-resistance, and virulence, and retrieved functional association between them. With complementation assays with top candidate genes, we could validate a gene for negative regulation of meropenem resistance and four genes for positive regulation of virulence in Galleria mellonella larvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antimicrobial resistance and virulence of human pathogenic bacteria with genomic and transcriptomic profiles from antibiotic-resistant clinical isolates.
Project description:The objective of this study was to analyse proteomic profiles of antibiotic-susceptible E. coli MG1655, K. pneumoniae NCTC418, E. faecium NCTC13169, and S. aureus NCTC8325 under sub-inhibitory antibiotic stress. Gram-negative bacteria were exposed to cefotaxime, ciprofloxacin, kanamycin, and imipenem, while Gram-positive bacteria were treated with chloramphenicol, vancomycin, or oxacillin.
